THE L-XYLULOSE-XYLITOL ENZYME AND OTHER POLYOL DEHYDROGENASES OF GUINEA PIG LIVER MITOCHONDRIA* BY SIEGFRIED HOLLMANNt AND OSCAR TOUSTER (From the Department of Biochemistry, Vandwbilt University School of Medicine, Nashville, Tennessee) (Received for publication, July 30, 1969) The mitochondria of guinea pig liver contain an enzymatic system which can reduce L-xylulose, the characteristic sugar in the urine of pento- Downloaded from www.jbc.org by guest, on March 26, 2013 suric individuals, to xylitol (2). In view of the possible importance of this system in the normal metabolism of the ketopentose, further investi- gation has been made of the transformation and of its possible relationship to other enzymatic reactions. We reported briefly (3) that a preparation obtained from the insoluble portion of ruptured guinea pig liver mitochon- dria contains at least two distinct polyol dehydrogenases (“ketose reduc- tases”) : (1) a triphosphopyridine nucleotide (TPN)-dependent enzyme which catalyzes the interconversion of n-xylulose and xylitol, and (2) a diphosphopyridine nucleotide (DPN)dependent system which acts on n-xylulose and xylitol as well as on several other ketoses and polyols. The present paper describes the preparation of these enzymes in soluble form and characterizes them in regard to substrate specificity, stability, and other properties. EXF’ERIMENTAL Materials-L-Xylulose was prepared by isomerization of L-xylose in pyridine (2). n-Xylulose was obtained from n-xylose by a similar pro- cedure . Xylitol was prepared by refluxing n-xylose for 7 hours in 166 parts of 70 per cent ethanol containing 10 parts of Raney nickel (aged 5 months) (4). The filtered solution was evaporated, under vacuum, to a green syrup which was extracted with boiling absolute ethanol. Evapora- tion of the extract yielded a light yellow syrup which crystallized from ethanol. Three recrystallizations from ethanol and, finally, one from methanol gave a total yield (including fractions from mother liquors) of 36 per cent, m.p. 91.5-92.5’. Dr. N. K. Richtmyer, of the National In- * This study was supported by a grant from the National Science Foundation. A preliminary account wac presented before the Forty-seventh annual meeting of the American Society of Biological Chemists at Atlantic City, April, 1956 (1). t Fulbright Scholar, 19554%; aided by a grant from the United States Department of State. Permanent address, Phyeiologisch-chemiechea Institut der Universitlit, Gattingen, Germany. 87 88 L-XYLULOSE-XYLITOL ENZYME stitute of Arthritis and Metabolic Diseases,generously furnished n-threitol, n-arabitol, n-talitol, n-gulitol, n-iditol, D-glycero-D-mannoheptitol (volemi- tol) , D-gly Cero-D-glUcOheptitO1 @-sedoheptitol) , L-glycero-n-mannoheptitol (perseitol), D-sorbose, n-iditol hexaacetate, and cY-D-altroheptulose hexa- acetate. The D-iditol derivative was catalytically deacetylated at room temperature with barium methoxide. Since in this procedure a good crystalline product was not obtained, paper chromatographic assay was employed to estimate the amount of the desired polyol in the preparations tested. The deacetylation of the altroheptulose derivative was carried out with barium methoxide at 0”. Paper chromatography indicated that a high yield of sedoheptulose was obtained. The following substrates were kindly contributed by other investigators: Downloaded from www.jbc.org by guest, on March 26, 2013 n-ribulose (as the o-nitrophenylhydrazone), Dr. B. L. Horecker, of the National Institute of Arthritis and Metabolic Diseases; L-ribulose, Dr. W. A. Wood and Dr. F. Simpson, of the University of Illinois; n-fuculose (as the o-nitrophenylhydrazone), Dr. M. Greene and Dr. S. S. Cohen, of the Children’s Hospital of Philadelphia; L-erythrulose, Dr. G. C. Mueller, of the University of Wisconsin; L-mannitol, Dr. E. Baer, of the Banting Institute. All other substrates were commercial samples. Erythritol, L-arabitol, and dulcitol were obtained from the Mann Research Labora- tories, Inc.; n-mannitol (mannite), from Merck and Company, Inc.; ribitol (adonitol), from the Nutritional Biochemicals Corporation; n-sorbitol, from Matheson, Coleman, and Bell; n-fructose, from the Difco Laboratories, Inc.; n-sorbose, from the Pfanstiehl Chemical Company; allitol, n-manno- heptulose, and n-glucoheptulose, from General Biochemicals, Inc. DPN, DPNH, and TPN were products of the Pabst Laboratories and of the Sigma Chemical Company. These coenzymes were at least 90 per cent pure. Muscle lactic dehydrogenase was purchased from the Worth- ington Biochemical Corporation, and spinach chloroplast TPN- diaphorase was a gift from Dr. Andre Jagendorf of the McCollum-Pratt Institute, The Johns Hopkins University. According to Dr. Jagendorf, 1 unit of enzyme is the amount causing a change in optical density of 1.0 Beckman unit per minute at 620 mp with 2,3,6-trichlorophenol-indophenol as the electron acceptor. He advised us that 1 unit per 3.0 ml. of reaction mix- ture would be an ample excess of enzyme. Methods-All spectrophotometric measurements were made with a Beckman model DU spectrophotometer. Unless otherwise indicated, sub- strate specificity tests were carried out at 23” in quartz cuvettes having approximately 3 ml. capacity and a 1.00 cm. light path. Contents of a typical test solution (3.0 ml. total volume) were as follows: 0.3 ml. of 0.5 M tris(hydroxymethyl)aminomethane (Tris) buffer at pH 8.1, 0.3 ml. of 0.08 M MgCh, 0.3 ml. of enzyme extract, 0.392 pmole of coenzyme, and S. HOLLMANN AND 0. TOUSTER 89 19.7 pmoles of polyol or 6.66 pmoles of ketose. When several substances were tested during an experiment, they were usually added in 0.05 ml. of water in order to minimize changes in concentration of components already present. Total volumes of 1 ml. were used for a few substrates available in very limited amounts, all components being reduced proportionately. Readings were made against solutions containing all components except substrate. Color reactions used for identification of ketoses were (1) the cysteine- carbazole reaction (5)) (2) the orcinol reaction (6), with a 40 minute heating period, and (3) a cysteine-sulfuric acid reaction kindly furnished by Dr. G. Ashwell of the National Institute of Arthritis and Metabolic Diseases.’ Before carrying out the various calorimetric analyses, reaction mixtures Downloaded from www.jbc.org by guest, on March 26, 2013 were deproteinized by addition of 2 volumes each of 5 per cent ZnSO1.- 7HzO and 0.29 N Ba(OH)2 and of 5 volumes of water, the reagents having been adjusted in strength to yield a final pH of approximately 7.2. Preparation of Enzyme-The entire procedure was carried out at O-2” except when otherwise indicated. The liver mitochondria of fasted guinea pigs (Carworth Farms, Inc.) were prepared as described previously (2), except that the mitochondrial pellet was washed three times with 0.15 M KCl-0.01 M NaHC03. For the mitochondria from 30 to 40 gm. of liver, the volumes of these successive washes were 180 ml., 120 ml., and, finally, a volume equal to 53 per cent of the weight of the liver used (yielding a suspension equivalent to a “65 per cent homogenate” of the liver). After the last centrifugation (10 minutes, 3400 r.p.m., International PR-1 cen- trifuge, rotor No. 823), the pellet was suspended in the “53 per cent” volume of water. The suspension was allowed to stand for 1 hour, and then the rupturing of the mitochondria was completed with a motor-driven homogenizer composed of a Teflon pestle and smooth glass tube. Mito- chondria could no longer be detected by phase microscopy. The insoluble portion of the ruptured particles was collected by centrifugation at 10,000 r.p.m. for 10 minutes in the high speed attachment (rotor No. 296) of the International centrifuge. The precipitate (the “mitochondrial residue”) was at times tested directly, in which case it was suspended in the 53 per cent volume of water. In other experiments the precipitate was washed with water or with phosphate buffer prior to testing its activity. To liberate the dehydrogenases, the residue was subjected to a modifica- tion of Morton’s butanol procedure for the isolation of succinic dehydro- genase (7). In a typical experiment, the mitochondrial residue from 43 gm. of liver was suspended in 25 ml. of 0.02 M sodium phosphate buffer at pH 7.88, the total volume being 33.5 ml. The suspension was cooled to -4’to -5’, and 13.4 ml. (40 per cent of the volume of the residue suspen- l To be published. 90 L-XYLULOSE-XYLITOL ENZYME sion) of n-butyl alcohol were slowly added, with continuous stirring, from a capillary burette. The addition was carried out over a period of at least 30 minutes. An addition time of 1 to 2 hours seems to enhance recovery of most of the dehydrogenases, but a specific study of this variable was not made. The mixture was stirred for 30 minutes after completion of the butanol addition. The emulsion was centrifuged at 19,000 r.p.m. for 30 minutes, the butanol layer was decanted, the mat of denatured protein was removed with a spatula, and the clear, yellow aqueous layer filtered into a flask for lyophilization. The primary purpose of the lyophilization is to remove the butanol, since dialysis inactivates the TPN enzyme. The dried product can be dissolved in water and filtered to yield a solution which is stable for many weeks in the refrigerator. Downloaded from www.jbc.org by guest, on March 26, 2013 Results Properties of Mitochmdrial Residue-Practically all of the activity of the mitochondria towards L-xylulose is present in the insoluble portion of the particles, the supernatant solution after centrifugation having negli- gible action on the pentose. However, if the residue iswashed with water, it is no longer active in the presence of the usual constituents of the incuba- tion medium, namely, phosphate buffer, MgCh, and glutamate. (Adeno- sine triphosphate was also added because it had been required in early mitochondrial experiments (2).) On the assumption that the washing process removes soluble glutamic dehydrogenase which adheres to the precipitate, DPNH was substituted for the glutamate. Fig. 1 shows that this replacement led to utilization of the ketose. The interpretation of this experiment is not clear. Addition of the soluble mitochondrial portion plus glutamate did not permit xylulose utilization by the washed residue or by enzyme rendered soluble. Furthermore, as will be evident below, relatively little of the L-xylulose enzyme can be obtained in soluble form if the residue is washed with water preliminary to the butanol treatment. Nonetheless, the experiments with washed residue and DPNH led to em- phasis on the pyridine nucleotide requirement and therefore facilitated initial studies on the soluble extract. Some experiments on the mitochondrial residue (unwashed) indicated that (1) Tris buffer and phosphate buffer are equally useful, (2) the optimal pH for L-xylulose disappearance is approximately 7.5, and (3) magnesium chloride and glutamate are required for maximal activity. The mito- chondrial residue could convert xylitol to ketopentose to a small extent (5.8 per cent) if DPN and methylene blue were added to the usual medium at pH 7.42. However, increasing the alkalinity was more effective than addition of coenzyme and dye in reversing the reaction. A 15 per cent yield was obtained in 90 minutes in a flask containing 0.1 ml. of 0.5 M Tris S. HOT&MANN AND 0. TOUSTER 91 buffer at pH 9.0,O.l ml. of 0.08 M MgCh, 20.7 pmoles of DPN, 19.9 ccmoles of xylitol, and 0.2 ml. of a suspension of mitochondrial residue (in 0.02 M phosphate buffer at pH 7.9) equivalent to a 50 per cent homogenate of whole liver in a total volume of 1.0 ml. The ketopentose was determined 0 SOL. EXTRACT f DPNH Downloaded from www.jbc.org by guest, on March 26, 2013 TIME IN MINUTES TIME IN MINUTES Fxa. 1 FIQ. 2 FIG. 1. Utilization of L-xyluloae by insoluble portion of ruptured mitocbondria. Each flask contained 0.3 ml. of 0.5 M glutamate, 0.9 ml. of 0.154 M phosphate buffer at pH 7.4, 0.3 ml. of Tris buder at pH 7.60.3 ml. of 0.08 M MgCL, 0.6 ml. of a sus- pension of residue in the 53 per cent volume of water, and 26 pmoles of xylulose. ATP ‘was added as 0.3 ml. of a 0.06 M solution; total volume, 3.0 ml. The flask in the DPNH trial contained 26.6 gmoles of NazDPNH and 13.3 &moles of L-xylulose in a total volume of 2.0 ml., all other components being reduced proportionately. The washed residue was prepared by twice suspending a portion of a residue preparation in the 53 per cent volume of water and collecting it each time by centrifugation for 10 minutes at 16,OW r.p.m. Analysis of Ba-Zn filtrates by the cysteine-carbazole method. FIG. 2. Utilization of L-xylulose by enzyme solution obtained from mitochondrial residues. The flask with residue contained 0.3 ml. of 0.5 M glutamate, 0.9 ml. of 0.154 M phosphate buffer at pH 7.4, 0.3 ml. of 0.03 M MgCL, 0.6 ml. of residue in the 53 per cent volume of water, 0.3 ml. of the soluble portion of ruptured mitochondria, and 20 pmoles of xylulose; total volume, 3.0 ml. (The soluble mitochondrial fraction was later found to be unnecessary.) The extract waz tested in a solution containing 0.3 ml. of 0.154 M phosphate buffer at pH 7.4,0.1 ml. of 0.03 M MgCl2, 13.3 pmoles of NazDPNH, 0.2 ml. of enzyme extract, and 6.66 pmoles of xylulose; total volume, 1.0 ml. Analysis of Ba-Zn filtrates by the cysteine-carbazole method. in Ba-Zn filtrates by the cysteine-carbazole method. The residue had no action on n-erythrulose, n-sorbose, or n-arabitol. Soluble n-Xylulose-Xylitol System-Fig. 2 shows that extraction of the n-xylulose-reducing enzyme is accomplished in high yield. It should be noted that the ratio of DPNH to n-xylulose in this experiment was 2: 1. Early in the work with the soluble enzyme solution it was found that, if the coenzyme is limiting (xylitol to coenzyme ratio of 50: l), TPN is much 92 L-XYLULOSE-XYLITOL ENZYME more effective than DPN in promoting the dehydrogenation of xylitol. It was tentatively assumed that the enzyme resembled other infrequently encountered enzymes which are not very specific in their coenzyme require- ment. However, tests of ketose reduction in the presence of limiting amounts of the reduced coenzymes indicated clearly that they act in dif- ferent enzymatic reactions. DPN is involved in the interconversion of xylitol and n-xylulose and of several other polyols and their corresponding ketoses. TPN, on the other hand, is the coenzyme in the interconversion of xylitol and L-xylulose by an enzyme which has unique substrate speci- ficity., With TPN (or TPNH for ketoses), the following compounds are practically inactive as substrates: L-threitol, erythritol, ribitol, D-arabitol, L-arabitol, D-sorbitol, dulcitol, n-mannitol, L-mannitol, D-talitol, n-gulitol, Downloaded from www.jbc.org by guest, on March 26, 2013 D-idikd, L-iditO1, ditO1, D-glyCerO-D-~~OheptitOl, D-&CerO-D-glUCO- 0 40 00 120 160 200 240 TIME IN MINUTES Fro. 3. Substrate specificity test of TPN system. Procedure as described under “Methods” (3 ml. cells). heptitol, L-glycero-n-mannoheptitol, L-erythrulose, D-ribulose, L-ribulose, n-xylulose, n-fructose, n-sorbose, L-sorbose, L-fuculose, sedoheptulose, n-mannoheptulose, and n-glucoheptulose. Only the n-gulitol could be considered to have slight activity; this indication may have been due to a trace of impurity. A typical substrate specificity test is illustrated in Fig, 3. The influence of pH on the equilibrium point is shown by the ef- fects of the HCl additions in lowering the final concentration of TPNH. Except for L-xylulose, no ketose caused a decrease in absorption at 340 rnp. The identification of the xylitol product as xylulose has already been reported (3), the evidence having been based upon absorption spectra of the colors produced in the orcinol and in the cysteine-carbazole reactions and on the rate of color formation in the latter test. Efforts were made to increase the yield of ketose. The first experiment f We suggest that this enzyme be named I%-xylulose (xylitol) reductase.” The existence of the two xylitol enzyme systems reported in this paper, as well as our recent finding (unpublished) of L-arabitol, perhaps derived from L-xylulose, in pentosuric urine, makes it desirable to include both the ketose and polyol in the name. S. H.OLLMANN AND 0. TOTJSTER 93 involved the use of borate, but 20 pmoles of this agent, added at equilib- rium to the regular incubation mixture, increased the TPNH concentra- tion by only a small amount. Even a solution containing Tris buffer at pH 9.0, MgCL, enzyme extract, and a xylitol to TPN ratio of 1: 1.3 gave a yield of only 2.7 per cent (by cysteine-carbazole analysis after Ba-Zn deproteinization). Dr. Jagendorf’s TPN-diaphorase was then used in an attempt to increase the yield. One experiment with 2,6dichlorophenol- indophenol was unsuccessful, but methylene blue proved useful. For one methylene blue trial the initial composition of the incubation mixture was as follows: 0.50 ml. of 0.5 M Tris buffer at pH 8.12,0.50 ml. of 0.08 M MgCh, 1.0 ml. of guinea pig enzyme solution, 329 moles of xylitol, 2 units of TPN-diaphorase, 16.44 pmoles of TPN, and 20.5 pmoles of methylene Downloaded from www.jbc.org by guest, on March 26, 2013 blue in a total volume of 2.8 ml. Two further additions of enzymes and water were made during the 25 hour incubation at 21-23’ (with occasional shaking of the flask). The 6nal amounts were for guinea pig enzyme, 2.0 ml.; diaphorase, 6 units; total volume, 5.0 ml. The yield of ketopentose was 5.8 per cent. A similar experiment involved the use of 12 units of the diaphorase, 32.86 pmoles of TPN, and an incubation for 28 hours at 24-26”. The yield was 7.2 per cent. It has been shown that equal amounts of xylulose and TPNH are formed in a solution containing buffer, MgCh, xylitol, and TPN (3). It was therefore possible to determine, without directly measuring the amount of xylulose formed, the equilibrium constant for the reaction Xylitd + TPN+ S L-xylulose + TPNH + H+ The enzyme preparation used in determining the constant had been heated at 50’ for 20 minutes, a treatment which usually causes a slight increase in activity. With incubation mixtures initially containing 22.97 pmoles of xylitol, 0.4576 Ltmole of TPN, 0.35 ml. of 0.5 M Tris buffer, 0.35 ml. of 0.08 M MgCh, and 0.35 ml. of heated enzyme solution in a total volume of 3.50 ml., and with final pH values of four flasks varying from 6.96 to 8.70, an average K of 8.58 X lo+ M was obtained after the 4 hours required for equilibrium to be established. Other Properties of TPN System-An experiment has been reported which shows that little L-xylulose enzyme is obtained if the mitochondrial residue is washed three times with water prior to the butanol treatment (3). Ap- proximately 40 per cent of the activity is lost if the residue is washed twice with 0.02 M phosphate buffer at pH 7.88 prior to extraction of enzyme. This is in contrast to the effect on the DPN-dependent enzymes, which are increased in yield (or activity) if the residue is first washed with either water or phosphate (see below). Another property of the L-xylulose enzyme by which it can be contrasted with the other dehydrogenases is its ready inactivation by dialysis. Reac- tion between TPN and xylitol could not be detected with enzyme which 94 L-XYLULoSE-XYLITOL ENZYME had been dialyzed against water (3 ml. of extract, 2 liters of water at 2”, 16 hours of dialysis). Even with use of the procedure of Nicholas and Nason (8), in which the dialysis membrane is first soaked in 0.15 M phos- phate at pH 7.4 containing lo-’ M glutathione and the dialysis is then carried out against 0.10 M phosphate containing 5 X 10-* M cysteine, only 37 per cent of the activity of undialyzed enzyme was obtained. The dial- ysis time was 16 hours. In spite of the lability of the enzyme to dialysis and to washing of the mitochondrial residue, it is stable in aqueous solution for many weeks at 2’ and for 20 minutes at 50”. It is stable at pH 4 for 2 hours at 2’ and, as many long spectrophotometric experiments show clearly, at pH 9 for sev- eral hours at room temperature. Downloaded from www.jbc.org by guest, on March 26, 2013 The xylitol-L-xylulose interconversion catalyzed by the soluble enzyme preparation is almost completely inhibited by 0.004 M iodoacetate, a finding in accord with the results obtained with intact mitochondria (2). Amino- pyrine, a weak inhibitor of the mitochondrial system (2), had no effect at a concentration of 0.02 M. Although magnesium chloride was always in- cluded in the medium, the question of its essentiality is still undecided. Magnesium ions were clearly necessary for full activity of particulate prep- arations. With enzyme rendered soluble, magnesium chloride had a small stimulatory effect and 0.002 M sodium versenate caused only a little in- hibition. 0.003 M dinitrophenol was without effect. for Substrates DPN-Dependent Tranqfomatims- Evidence for the exist- ence of more than one mitochondrial polyol dehydrogenase was obtained when the enantiomorphic forms of xylulose were tested in incubation mix- tures containing DPNH and TPNH formed by the dehydrogenation of xylitol. With DPNH, L-xylulose had little effect, but n-xylulose caused a rapid decrease in concentration of the reduced coenzyme. Studies men- tioned above showed clearly that the DPN and TPN enzyme systems dif- fered in their response to various treatments.’ The substrate specificity of the DPN system was then studied with the view towards obtaining a more complete characterization, especially in relation to the “soluble” (non- particulate) liver polyol dehydrogenase of Blakley (9), which catalyzes the interconversion of xylitol and D-xylulose (10). Table I summarizes these substrate specificity tests. The typical behavior of several active sub- strates is shown in Fig. 4, together with the inhibitory effect of 0.004 M iodoacetate. The lack of substrate activity of L-xylulose, L-sorbose, and D-fructose (all used in 6.66 eole amounts) and the rapid reduction of D-xylulose and of n-ribulose were shown directly in experiments with crys- 8 The DPN and TPN systems have recently been separated from each other (FL Wohl and 0. Touster, unpublished results). S. HOLLMANN AND 0. TOUSTER 95 talline DPNH. In these experiments, readings were made against solu- tions containing substrate but no coenzyme. Corrections were made for TABLE I &&i&f/ of DPN &/Ste??Z Polyols I Ketoses Indicated reaction Substrate eactivitj Substrate leactivity n-Threitol + n-Erythrulose + L-Threitol G= L-eryth- rulose Erythritol Xylitol D-Xylulose + Xylitol * D-xylulose Downloaded from www.jbc.org by guest, on March 26, 2013 Ribitol : D-Ribulose + Ribitol ti D-ribulose D-Arsbitol n-Ribulose n-Arabitol n-Xylulose D-Sorbitol + D-Fructose 4-* D-Sorbitol C= D-fruc- tose D-Iditolt D-Sorbose n-Iditol + t-Sorbose -I-* L-Iditol C= L-sorbose Dulcitol - D-Mannitol - n-Maxmitolt D-Talitol - D-Gulitol Allitolt n-Fuculoset D-Glycero-D-gluco- + Sedoheptuloset D-Glycero-D-glucO- heptitol heptitol c= L-gulo- heptulose (?)$ n-Glycero-D-man- D-Mannoheptulose noheptitol D-Glycero-D-man- D-Glucoheptuloset noheptitol * Activity could be detected only when used at concentrations 24 times that used with other active ketoses (see under “Methods” and “Hexitol-ketohexose trans- formations”). t 1.0 ml, reaction mixtures were used in these cases. All other substances were tested in standard 3 ml. procedure described under “Methods.” $ It is possible that sedoheptulose is the ketose reactant and that, owing to an especially low equilibrium constant, a very high concentration would be required to show its activity. the slow decrease in DPNH concentration which occurred in the absence of a reactive ketose. X@itoCD-X&lose Interwnversion -Our previous report (3) presented evidence that the dehydrogenation product of xylitol in the presence of 96 L-XYLULOSE-XYLITOL ENZYME DPN is xylulose and that equal amounts of the ketopentose and reduced coenzyme are produced. Equilibrium constants at various pH values were determined as described above for the xylitol-L-xylulose transformation, except that DPN was substituted for the TPN and that five flasks with final pH values varying from 7.01 to 8.76 were employed. An average K of 1.55 X lo-l1 M was obtained. Efforts to increase the yield of ketopentose were based on coupling of the transformation to pyruvate with lactic dehydrogenase. Two different batches of the polyol dehydrogenase gave xylulose yields of 2.5 and 5.9 per cent, respectively. In the latter experiment the incubation mixture had the following initial composition: 0.5 ml. of 0.5 M Tris buffer at pH 8.12, 0.5 ml. of 0.08 M MgCh, 1.0 ml. of guinea pig enzyme solution, 329 pmoles Downloaded from www.jbc.org by guest, on March 26, 2013 TIME IN MINUTES FIQ. 4. Activity of DPN system in interconversion of ribitol and n-ribulose and of xylitol and n-xylulose; inhibition of ketopentose reduction by iodoacetate. Pro- cedure as described under “Methods” (3 ml. cells). The sodium iodoacetate was added as 0.22 ml. of a 0.06 M solution. of xylitol, 0.2 ml. of a 1:250 dilution of Worthington lactic dehydrogenase in 0.01 M NaCl, 32.86 pmoles of DPN, and 300 pmoles of sodium pyruvate in a total volume of 3.0 ml. During the 28 hour incubation at 24-26” (with occasional shaking of the flask), two 0.5 ml. additions of guinea pig enzyme and five 0.2 ml. additions of lactic dehydrogenase solution were made. In the former experiment, in which the lower yield was obtained, only half these amounts of lactic dehydrogenase and DPN were employed. The xylitol-n-xylulose enzyme system was usually activated somewhat by preliminary washing of the mitochondrial residue (see below) and by dialysis. Like the L-xylulose enzyme, it is stable between pH 4 and 9 and is inhibited by 0.004 M iodoacetate (Fig. 4). Rib&l-n-Ribulose TransforrnatimL-It was of interest to characterize the product of the DPN-ribitol reaction. A reaction mixture similar to the one employed with xylitol (3) was used, 90 mg. of charcoal (Darco G-60) 8. HOLLMANN AND 0. TOUSTER 97 per 1.8 ml. of Ba-Zn filtrate being required to yield a DPN-free solution for the orcinol determination. The peaks of the absorption spectrum of the color produced had the following ratios: 540:670 nn~, 0.75; 435~670 mp, 0.63. Authentic ribulose gave ratios of 0.75 and 0.66, respectively. Both the ribitol product and ribulose showed the same rapid attainment of maximal color in the cysteine-carbazole test (less than 10 minutes). Coupling of the ribitol dehydrogenation to lactic dehydrogenase and sodium pyruvate, with the same procedure as was used with xylitol, gave keto- pentose yields of 3.2 and 5.4 per cent, the comparable values for xylitol being 2.5 and 5.9 per cent, respectively. The similar yields of xylulose and ribulose from the corresponding pentitols might suggest that the same Downloaded from www.jbc.org by guest, on March 26, 2013 0 40 80 120 0 20 40 60 00 TIME IN MINUTES FIG. 5. Reaction of DPNH with relatively high concentrations of n-fructose and L-sorbose. Procedure aa described under “Methods” (3 ml. cells). Unless other- wise indicated, the amounts of polyols and ketoses added were 19.7 and 6.66 pmoles, respectively. enzyme was catalyzing the two reactions. However, not all enzyme preparations had similar ratios of activity towards the two pentitols. With all enzyme preparations, xylitol and n-xylulose reacted more rapidly than did ribitol and n-ribulose, respectively. Fig. 4 shows a comparison of the two pentitol reactions and the inhibition of n-ribulose reduction by iodo- acetate. Hex&d-Ketohexose Transforma2ionsThese reactions presented a prob- lem because the usual spectrophotometric assay procedure failed to show activity of the ketohexoses expected to be produced from n-sorbitol and from L-iditol (Fig. 5, A). n-sorbitol was an active substrate of all enzyme preparations; yet n-fructose and L-sorbose,the two possibleproducts (assum- ing dehydrogenation of the second or fifth alcohol grouping), showed no activity under the usual very favorable conditions for ketopentose reduc- 98 L-XYLULOSE-xmIT0L mznm tion. L-Sorbose is also the expected product from n-iditol. It was there- fore necessary to characterize the dehydrogenation products by various calorimetric and paper chromatographio methods. That fructose is indeed produced from D-sorbitol is indicated by the fol- lowing evidence. Firstly, the sorbitol product and fructose gave similar colors in the cysteine-carbazole test (565 rnp peak), in the orcinol reaction (Fig. S), and in the cysteine-sulfuric acid test (Fig. 6). The colors in the latter two tests were unlike the colors given by sorbose (Fig. 7). Secondly, paper chromatographic comparison, with 80 per cent n-propyl alcohol as solvent and naphthoresorcinol spray (ll), showed identical Rp values and Downloaded from www.jbc.org by guest, on March 26, 2013 WAVE LENGTH (mp) WAVE LENGTH (mp) FIG. 6 FIG. 7 FIG. 6. Absorption spectra of h-fructose and of the u-sorbitol product in color tests. 0, u-sorbitol product; 0, u-fructose, in the orcinol reaction; A, u-sorbitol product; A, D-fructose, in the cysteine-sulfuric acid reaction. FIG. 7. Absorption spectra of L-sorbose and of the L-iditol product in color tests. l , L-iditol product; 0, L-sorbose, in the orcinol reaction; A, L-iditol product; A, L-sorbose, in the cysteine-sulfuric acid reaction. color development. Thirdly, inclusion of n-fructose in two incubation mixtures caused a decrease in rate of DPNH formation, from sorbitol and DPN, proportionate to t,he fructose concentration. Finally, by use of a very high concentration of n-fructose (24 times the effective ketopentose level), it was possible to show that the ketohexose can effect the slow oxida- tion of DPNH (Fig. 5, B). The L-iditol reaction was studied similarly. Fig. 7 shows that the reac- tion product and sorboseyield similar colors in the orcinol reaction and in the cysteine-sulfuric acid test. In the cysteine-carbazole test, identical spectra and rates of color development were obtained. Furthermore, pa- per chromatographic comparison (80 per cent n-propyl alcohol solvent and naphthoresorcinol spray) provided additional confirmation. As with n-fructose, the use of large amounts of n-sorbosefinally disclosed its activity S. HOLLMANK AND 0. TOUSTER 99 as a substrate (Fig. 5, B). The inactivity of a similar concentration of n-sorbose is shown for comparison. Multiplicity of DPN-Dependent Polyol Dehydrogenases-Evidence for the presence of more than one DPN-dependent polyol dehydrogenase in mito- chondria was provided by the observation that dehydrogenation rates for several polyols varied unpredictably with various enzyme extracts. While our earlier extracts showed greater activity towards xylitol than towards all other polyols, more recent ones have been at least as active towards n-sorbitol as towards xylitol. Comparison of polyol oxidation rates of sev- eral extracts is shown in Table II. Apparent similarities among the DPN enzymes may be due to secondary factors. Fig. 8 shows that extracts prepared from mitochondrial residues Downloaded from www.jbc.org by guest, on March 26, 2013 which have been washed with phosphate buffer have enhanced activity TABLE II Relative Rates of Reaction of Xylitol, D-Sorbitol, and L-Iditol in DPN System* Enzyme preparation Substrate 16C If A 18A 19A 20A __~-~-~~ Xylitol.......................... 0.024 0.048 0.029 0.024 0.040 D-Sorbitol.. 0.010 0.016 0.034 0.039 0.045 L-Iditol .. 0.022 0.057 * The values in the table are the increases in absorption at 340 rnp, during the 10 minute period following addition of substrate, of 3 ml. test solutions prepared aa described under “Methods.” (with DPN) towards xylitol and D-sorbitol, although the TPN-xylitol reac- tion rate is decreased. It is possible that a factor involved in DPN de- struction is removed in the washing process (or by dialysis, which usually activates the DPN-xylitol reaction). It may be mentioned here that wash- ing of the mitochondrial residue with water also activates the DPN-xylitol system although the TPN-xylitol reaction is decreased even more markedly than when phosphate is used (3). DISCUSSION It is necessary to emphasize that, in all the work prior to the studies on the soluble preparations, attention was paid almost exclusively to activity towards n-xylulose. In fact, under the ordinary test conditions, particu- late preparations showed no activity towards any compounds other than n-xylulose and xylitol. Even n-xylulose, a very active substrate with the aqueous extract, was not acted upon by mitochondria under conditions 100 L-XYLULOSE-XYLITOL ENZYME which led to reduction of L-xylulose (2). It is therefore possible that polyol dehydrogenases other than the ones reported are present in mitochondria but have not been examined appropriately, perhaps being inactivated or removed at some step in the fractionation. The lack of activity of D-xy- lulose with mitochondria may be due to the association of the xylitol-n- xylulose enzyme with an inhibitor which is removed during the prepara- tion of extract. The increased yield of the n-xylulose enzyme from washed mitochondria and the activation of extracts by dialysis provide some sup port for this explanation. Downloaded from www.jbc.org by guest, on March 26, 2013 20 40 0 20 49 0 20 40 TIME IN MINUTES FIQ. 8. Change in soluble polyol dehydrogenase activity resulting from washing of mitochondrial residues with phosphate buffer. A preparation of residue was divided into two portions. One was suspended in phosphate buffer, and butanol was added as described under “Preparation of enzyme.” The other portion was suspended twice in the proportionate volume of phosphate buffer and sedimented each time by centrifugation for 10 minutes at 16,000 r.p.m. The precipitate was then resuspended in buffer and subjected to the butanol procedure. l , extract ob- tained from unwashed residue; 0, extract obtained from washed residue. Although our DPN system differs in cellular location from that of Blak- ley’s enzyme, similarities in substrate specificity (10) led to concern about the possible identity of the two systems. Three differences in substrate reactivity are now evident: (1) n-fructose and L-sorbose are much less reactive with our extract than with Blakley’s rat liver extract; (2) sedo- heptulose, very active with a purified preparation from the soluble fraction of sheep liver (lo), is inactive with the extract derived from guinea pig liver mitochondria; (3) allitol, another substrate for the Blakley prepara- tion (lo), is inactive with ours. In retrospect it is realized that the ques- tion of identity of the mitochondrial and non-mitochondrial systems is probably a meaningless one, since it appears likely that both are mixtures of closely related enzymes. Evidence for the multiplicity of DPNdepend- ent polyol dehydrogenases in guinea pig liver mitochondria has already been presented in this paper. Williams-Ashman and Banks (12), who re- 5. HOLLMANN AND 0. TOUSTER 101 ported that the Blakley enzyme occurs also in the seminal vesicle and in the coagulating gland of the rat, found that L-arabitol is a substrate for a rat liver extract. This is in disagreement with another study (10). Fur- ther work is obviously necessary on the purification and characterization of the various liver polyol dehydrogenases. The equilibrium constants of both of the xylitol-xylulose reactions are considerably smaller than those reported for the sorbitol-n-fructose reac- tion catalyzed by t.he non-mitochondrial liver enzyme (9, 12) and for the mannitol-1-phosphate-n-fructose-6-phosphate reaction catalyzed by an en- zyme extracted from Escherichiu coli B (13). The guinea pig was originally chosen for the urinary L-xylulose studies because it resembled man in requiring L-ascorbic acid in the diet. Then, Downloaded from www.jbc.org by guest, on March 26, 2013 the liver of bhe guinea pig was used for the enzyme studies because the keto- pentose was found to be a constituent of its urine (14). These considera- tions may actually have had little influence on the progress of this investi- gation, but our few experiments on other species suggest that the Guinea pig was a fort,unat,e choice. Hamster, rat, and monkey liver preparations utilized L-xylulose more slowly than comparable ones from guinea pigs. The physiological significance of the mitochondrial dehydrogenases re- mains to be elucidated. Enzymatic activity of the kind reported here has not .been previously detected in mitochondria. Since the DPN system is unquestionably most active towards n-xylulose among the ketoses tested, and is also quite active towards xylitol, it is possible that the successive action of the two xylulose enzymes, both present in the same morphological unit, effects the interconversion of the enantiomorphic forms of the ketose. There is considerable evidence that n-glucuronolactone is a metabolic precursor of L-xylulose (14), but a normal role for the former substance has not been reported. Nevertheless, a substance derived from a glucuronic acid-containing metabolite may be the normal precursor of the pentose. The discovery of the key role of n-xylulose-5-phosphate in the 6-phospho- gluconate pathway suggests a route for L-xylulose to enter (or be formed from) a normal, major carbohydrate pathway. Yeast transketolase, re- ported recently to act on n-xylulose-5-phosphate rather than on D-ribulose- 5-phosphate, has some action on free n-xyluiose (15), but transketolase isolated from spinach is without action on the free pentose (16). A specific n-xylulose kinase from bacteria has been found which converts the pentose to its 5-phosphate derivative (17). The presence of a similar enzyme in liver would provide a bridge bet.ween the xylulose transformations and the 6-phosphogluconate pathway. Studies to detect such a kinase in liver have been initiated.4 4 While the present paper was in proof, Hickman and Ashaell (18) reported the occurrence of n-xylulokinase in calf liver. 102 L-XYLULOSE-XYLITOL ENZYME We acknowledge the able assistance of Mrs. Ruth Hutcheson Mayberry and Miss Margarita Escobedo G. in the preparation of xylulose, of Mr. John C. Kirschman in the preparation of xylitol, and of Mr. Richard Wohl in carrying out some substrate specificity tests. SUMMARY Studies on polyol dehydrogenases of the insoluble portion of ruptured guinea pig liver mitochondria are reported. The enzymes, made soluble by a modification of the butanol method, show activity towards a number of polyols and ketoses. Data presented indicate the presence of an en- zyme, unique among such dehydrogenases in its triphosphopyridine nu- cleotide (TPN) requirement, which catalyzes the interconversion only of Downloaded from www.jbc.org by guest, on March 26, 2013 xylitol and L-xylulose. The extract also contains diphosphopyridine nu- cleotide (DPN)-requiring dehydrogenases which catalyze the interconver- sion of several substrates, namely, xylitol and n-xylulose, L-threitol and n-er$thrulose, ribitol and n-ribulose, n-sorbitol and n-fructose, n-iditol and L-sorbose, and D-glycero-D-glucoheptitol and a heptulose. Evidence is pre- sented that the TPN- and DPN-dependent enzymes are distinct from each other, and the indications for the multiplicity of DPN-dependent liver polyol dehydrogenases are pointed out. The possibility that the mito- chondrial enzymes provide a connection between L-xylulose and the 6- phosphogluconate pathway is dis,cussed. BIBLIOGRAPHY 1. Touster, O., Reynolds, V. H., Hutcheson, R. M., and Hollmann, S., Federation PTOC., 16, 372 (1956). 2. Touster, O., Reynolds, V. H., and Hut,cheson, R. M., J. Biol. Chem., 221, 697 (1956). 3. Hollmenn, S., and Touster, O., J. Am. Chem. Sot., 78,3544 (1956). 4. Karabinos, J. V., and Ballun, A. T., J. Am. Chem. Sot., 76, 4591 (1953). 5. Dische, Z., and Borenfreund, E., J. Biol. Chem., 192, 583 (1951). 6. Mejbaum, W., 2. physiol. Chem., 258, 117 (1939). 7. Morton, R. K., in Colowick, S. P., and Kaplan, N. O., Methods in enzymology, New York, 1, 48 (1955). 8. Nicholas, D. J. D., and Nason, A., J. Biol. Chem., 207,353 (1954). 9. Blakley, R. L., Biochem. J., 49, 257 (1951). 10. McCorkindale, J., and Edson, N. L., Biochem. J., 67,518 (1954). 11. Bryson, J. L., and Mitchell, T. S., Nature, 187, 864 (1951). 12. Williams-Ashman, H. G., and Banks, J., Arch. Biochem. and Biophys., 50, 513 (1954). 13. Wolff, J. B., and Kaplan, N. O., J. BioZ. C&m., 218, 849 (1956). 14. Touster, O., Hutcheson, R. M., and Rice, I,., J. BioZ. Chem., 216, 677 (1955). 15. Srere, P. A., Cooper, J. R., Klybas, V., and Racker, E., Arch. Biochem. and Bio- phys., 69, 535 (1955). 16. Smyrniotis, P. Z., and Horecker, B. L., J. BioZ. Chem., 218, 745 (1956). 17. Stumpf, P. K., and Horecker, B. L., J. BioZ. Chem., 218,753 (1956). 18. Hickman, J., and Ashwell, G., J. Am. Chem. Sot., 78, 6209 (1956).
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